Peroxisome proliferator activator receptor- (PPAR) activation results in up-regulation of genes associated with skeletal muscle fatty acid oxidation and mitochondrial uncoupling. However direct, non-invasive assessment of lipid metabolism and mitochondrial energy coupling in skeletal muscle following PPAR stimulation has not been examined. Therefore in this study, we examined the response of a selective PPAR agonist (GW610742X at 5 or 100 mg/kg/day for 8 days) on skeletal muscle lipid metabolism and mitochondrial coupling efficiency in rats using in vivo magnetic resonance spectroscopy (MRS). There was a decrease in the intramyocellular lipid to total creatine (IMCL/tCr) ratio as assessed by in vivo ¹H MRS in soleus and tibialis anterior muscles by day 7 (49 and 46% respectively, P<0.01) at the high dose. Following the ¹H MRS experiment (Day 8), 1-¹³C glucose was administered to conscious rats in order to assess metabolism in the soleus muscle. The relative fat vs carbohydrate oxidation rate increased in a dose dependent manner (52% and 93% in the 5 and 100 mg/kg/day groups respectively, P<0.05). In separate experiments where mitochondrial coupling was assessed in vivo (Day 7), ³¹P MRS was used to measure hindlimb ATP synthesis and ¹³C MRS was used to measure the hindlimb tricarboxylic acid cycle flux (V_tca). There was no alteration, at either dose, in mitochondrial coupling efficiency measured as the ratio of unidirectional ATP synthesis flux to V_tca. Soleus muscle GLUT4 expression was decreased by 2 fold while PDK4, CPT1a, UCP2, and UCP3 expression was increased by 2-3 fold at the high dose (P<0.05). In summary, these are the first non-invasive measurements illustrating a selective PPAR mediated decrease in muscle lipid content which was consistent with a shift in metabolic substrate utilization from carbohydrate to lipid. However, the mitochondrial energy coupling efficiency was not altered in the presence of increased uncoupling protein expression.